This invention relates to a mixture of purified equine immunoglobulins suitable for use in treating failure of passive transfer in foals, and essentially free of non-immunoglobulin proteins, and to the methods of preparing and using it.
The newborn foal is very susceptible to infection immediately after birth. In nature, the foal is normally given protection by its mother's first milk, or colostrum, which is rich in maternal antibodies. The foal absorbs these antibodies during its first 24 hours of life. Afterward, the villous epithelial cells of the small intestine undergo changes which frustrate further adsorption of immunoglobulins.
Unfortunately, foals which are born too weak to nurse or which are rejected by their mothers after birth become immunodeficient. A complete or partial failure of passive transfer of immunity may also arise through premature lactation by the mare, abnormally low immunoglobulin content in the mare's colostrum, or abnormally poor absorption of immunoglobins by the foal. See Liu, Systemic Diseases of the Newborn Foal, Vet. Clinics of North Amer.: Large Animal Practice, 2: 361 (1980)
The treatment of failure of passive transfer in newborn animals by administration of colostrum milk or salt-purified Ig serum fractions is known. See Newson, U.S. No. 4,096,244; Line, U.S. No. 2,607,716; Heinbach, U.S. No. 3,128,230; Michaelson, U.S. No. 3,646,193; Khours, U.S. No. 3,984,539; Divers, et al., Am. Ass'n Equine Practitioners Proceedings, 473 (1982); Burton, et al., Am. J. Vet. Res., 42: 308 (1981); Le Blanc and Asbury, Equine Vet. Sci., 5: 78 (1983). Commercial preparations exist, notably FOAL IMMUNE (Lake Immunogenetics, 345 Berg Road, Ontario, N.Y. 14519) and EQUIGAM (Equigam Products, Inc., 1615 West Waters Ave., Tampa, Fla. 33604) which are merely sterile whole serum, containing antibodies. These crude preparations have numerous disadvantages, including short shelf lives, low immunologic potency, and contamination with antigenic or pyrogenic substances.
Perper, U.S. No. 3,664,994 used DEAE-dextran chromatography at pH 7.2-7.85 to separate equine immunoglobulins from serum. Procedurally, his method differed from that described herein in several respects: (1) use of DEAE rather than QAE; (2) use of a basic rather than an acidic buffer; and (3) retention rather than pass through of the antibodies. We have found that equine immunoglobulins, unlike human immunoglobulins, respond adversely to basic conditions. Our product also differs significantly from that of Perper, in that, to obtain an alleged pure equine Ig fraction, Perper limited himself to a recovery of 50 mg of protein from 25 ml of serum. It is believed that Perper's fractions do not contain all of the important equine antibodies, particularly anti-tetanus IgG-T, which is known to be difficult to purify. See Kent and Blackmore, Equine Vet. J. 17(2): 119-124 (1985). Perper was interested only in horse anti-lymphocyte gamma globulin, not in horse antibodies generally. We also believe that Perper's fractions may have been contaminated with transferrin, in view of our own experience with DEAE chromatography, as well as the reference to transferrin contamination in the manufacturer's literature regarding the use of Zetaprep-15 DEAE disks. It has been postulated that transferrin plays a role in iron toxemia in foals.
It is conventional in the art to purify immunoglobulins from sera by DEAE chromatography under basic conditions. See U.S. No. 4,541,953; U.S. No. 4,562,160; U.S. No. 4,331,649. Surprisingly, we discovered that use of such conditions in purifying equine Ig resulted in precipitation of the antibodies, followed by aggregate formation, when the product was concentrated or stored at 4.degree. C.
Equine antibodies have been studied to some degree. See Montgomery, Proc 3d Int. Conf. Equine Infectious Disease, Paris, 1972, at 343-63 (1973); McGuire, et al., Id., 364-81 (1973).
The major equine blood proteins are transferrin (a 90K beta-globulin), albumin, prealbumin, the XK protein family, hemoglobin and various enzymes. Braend, Id, 394-406 (1973). A purified Ig fraction should be free of these proteins.